Eukaryotic genomes are packaged in chromatin, a nucleoprotein complex containing DNA, histones, and nonhistone chromosomal proteins. The compaction thus provided presents a significant barrier to cellular processes such as replication and transcription that require access to template DNA. DNA viruses that infect eukaryotic cells have served as useful experimental subjects for studying effects of chromatin on gene regulation and replication. The VP16 protein of HSV-1 potently stimulates transcription of the viral immediate early genes and thus triggers the cascade of viral gene expression during the lytic infection cycle. In heterologous experimental contexts, VP16 can recruit chromatin modifying coactivators to target promoters. However, previous reports indicated that the large HSV-1 viral DNA genome remains predominantly non-nucleosomal during lytic infection. These observations reveal an apparent paradox: why would a viral activator such as VP16 recruit chromatin modifying coactivator proteins to a DNA template that lacks nucleosomes? The overall goal of this project is to define the roles of histones and chromatin in HSV-1 gene regulation, including the recruitment and activity of chromatin-modifying coactivator proteins on viral genes during infection. Our work to date has shown that certain coactivators do associate with viral DNA during HSV infection in a manner dependent on the VP16 activation domain. Moreover, histones are present at many regions of the viral DNA, but are absent from IE promoters in a VP16-dependent manner. In the proposed project period, the association of additional histones and nonhistone chromosomal proteins with HSV DNA throughout lytic infection will be tested. The presence of other coactivator proteins at IE promoters will be examined, as will the dependence of IE transcription on those coactivators. This work will advance the understanding of cellular regulatory mechanisms and the strategies used by HSV to adapt those mechanisms for viral -gene expression. Herpes simplex virus infections are widespread in the US population, and cause significant discomfort and occasional death. Early stages in virus infection seem to depend on certain enzymes from the host cell. This research will validate the roles of those enzymes in virus infection, which may lead to new antiviral drug targets.